Respiratory syncytial virus among hospitalized patients of severe acute respiratory infection in Bhutan: Cross‐sectional study

Abstract Introduction Respiratory syncytial virus (RSV) is a leading cause of lower respiratory tract infections worldwide, particularly in young children. In Bhutan, respiratory disease continues to be among the top 10 diseases of morbidity for several years. This study aimed to estimate the prevalence of RSV among hospitalized patients with severe acute respiratory infection (SARI) in Bhutan. Method Respiratory specimens were collected from SARI patients of all ages in 2016 and 2018 following influenza surveillance guidelines. Specimens were tested for influenza and RSV, human metapneumovirus, adenovirus, and human parainfluenza virus types 1, 2, and 3 using real‐time reverse‐transcription polymerase chain reaction assay. Descriptive statistics were used to analyze the result in STATA 16.1. Result Of the 1339 SARI specimens tested, 34.8% were positive for at least one viral pathogen. RSV was detected in 18.5% of SARI cases, followed by influenza in 13.4% and other respiratory viruses in 3%. The median age of SARI cases was 3 (IQR: 0.8–21 years) years. RSV detection was higher among children aged 0–6 (Adj OR: 3.03; 95% CI: 1.7–5.39) and 7–23 months (Adj OR: 3.01; 95% CI: 1.77–5.12) compared with the children aged 5–15 years. RSV was also associated with breathing difficulty (Adj OR: 1.73; 95% CI: 1.17–2.56) and pre‐existing lung disease, including asthma (Adj OR: 2.78; 95% CI: 0.99–7.8). Conclusion Respiratory viruses were detected in a substantial proportion of SARI hospitalizations in Bhutan.


| INTRODUCTION
Every year, millions of people around the world suffer from severe respiratory infections caused by a respiratory syncytial virus (RSV).This virus, first discovered in chimpanzees, belongs to the genus Orthopneumovirus within the family Pneumoviridae and order mononegavirales. 1,2V is a leading cause of severe respiratory infection causing hospitalization and death across all age groups, particularly among young children and older adults worldwide.4][5] Likewise, it is estimated that RSV causes about 1.5 million hospitalizations and 14,000 deaths annually among older adults in industrialized countries. 6,7re than 95% of RSV-ALRI episodes and more than 97% of RSV-attributable deaths across all age bands were in low-income and middle-income countries (LMICs). 80][11] Studies have shown that RSV infections have a seasonal pattern, with peaks occurring during winter months in temperate regions and during rainy seasons in tropical regions. 12,13wever, there is little or no data on the seasonality of RSV in Bhutan, which has a diverse climate ranging from subtropical to alpine. 14Moreover, there is a lack of information on the demographic and clinical characteristics of RSV patients in Bhutan, such as age, gender, comorbidities, symptoms, and outcomes. 157][18][19][20][21][22] Therefore, this study aims to determine RSV incidence among SARI cases, seasonal trends, risk factors, and outcomes for patients.Thus, the primary objective is to assess RSV association with demographic and clinical characteristics and to provide valuable information to inform public health strategies and clinical management of SARI in Bhutan.

| Study design and setting
We have adopted a prospective protocol design on existing national ILI and SARI sentinel-based surveillance for RSV pathogens as per the surveillance guidelines. 23Sentinel surveillance hospitals are Jigme Dorji Wanghck National Referral Hospital (JDWNRH), Thimphu, Punakha Hospital, Paro Hospital, Mongar Regional Referral Hospital, Phuentsholing Hospital, Chukha, Gelephu Regional Referral Hospital, Sarpang, Trongse Hospital, Tsirang Hospital, Trashigang Hospital, Samtse Hospital, and Samdrup Jongkhar Hospital (Figure 1).The sentinel sites were selected based on strategic geographical locations and demographic representation.surveillance guidelines. 23,24Nasal/throat swab specimens for molecular RT-PCR assays were collected as part of routine patient care through a surveillance network of the Inpatient Department or from the Hospital Emergency Department.

| Testing algorithm
The SARI specimens were received at the PCR lab in the Royal Centre for Disease Control (RCDC) from the SARI sentinel hospitals every week.The samples were first tested for influenza A and B by real-time RT-PCR.All influenza negative samples were further tested for other respiratory viruses (ORV) including RSV, hMPV, adenovirus, and parainfluenza 1, 2, and 3 as prescribed in the testing algorithm below (Figure 2).The cycling conditions were as follows: a single cycle of reversetranscription step at 50 C for 30 min, followed by an initial denaturation step at 95 C for 2 min.Subsequently, 45 amplification cycles were performed, consisting of denaturation at 95 C for 15 s and annealing/extension at 55 C for 30 s. Ct values were used to interpret the results.Ct values of <40 for influenza and RSV, and <35 for the human RNase P (RP) reaction, were considered positive, whereas Ct values of ≥40 for influenza and RSV, and ≥35 for RP, were considered negative.For each protocol, positive and negative controls were run in each test to validate the test result.

| Data collection
Demographic data including clinical and laboratory information were collected through a structured surveillance questionnaire of the SARI Specimen Collection Form (Figure A1), and information on the form was verified before entering into the surveillance system.All laboratory results for influenza and other respiratory viruses were entered into the system, and feedback was shared weekly (FluView Report) with all relevant stakeholders and sentinel sites. 25

| RESULT
A total of 1339 SARI cases were enrolled.The median age of SARI was 3 years (IQR: 0.8-21 years) and 56% were males.Virus detection was reported in 34.8% of tested specimens.RSV was detected in 18.5% (248/1339) of SARI cases, followed by influenza in 13.4% (180/1339) and ORV in 2.8% (38/1339).Demographic and clinical characteristics of RSV, influenza, and ORV-associated SARI cases are shown in Table 1.Among RSV-associated SARI cases, 86% were below 5 years of age and 52% were females.Among influenzaassociated SARI cases, 45.6% were below 5 years and 61% were males.Clinically, difficulty breathing was more prevalent in RSV-associated SARI (85%).ICU admission was observed in 6.6% of all enrolled cases and more common in RSV cases (8.2%) as compared with influenza and other viruses (Table 1).
RSV detection was seen throughout the year in 2016 and 2018 but had an observed in increase positivity rate (>10%) between January to April and June to September in both years (Figure 3).RSV and influenza were detected from all the SARI sentinel hospitals, though RSV positivity was high in JDWNRH, Thimphu, and Paro Hospital with 26% to 29.6% followed by Trongsa Hospital and Samtse Hospital with 17.3% to 26.0% (Figure 4).

| DISCUSSION
In this study, we found respiratory viruses were detected in one-third of SARI cases, and the most frequently detected virus was RSV followed by influenza.RSV was detected in nearly one in five of all SARI cases.However, we need to consider the SARI case definition, which does not meet the optimal criteria for RSV surveillance as per the WHO; therefore, the case definition needs to be expanded beyond SARI to include cases that do not have a fever or a history of fever.
Hospital-based RSV surveillance will use the extended definition of SARI, also hospital-based inpatient RSV surveillance in children aged 0-<6 months will additionally include those who present with apnea or sepsis (or both). 26Both RSV and influenza circulated throughout the year and had 2 peaks-one between January to April and a second between June to September.However, 2 years of surveillance might also be insufficient to determine seasonality.The same two peak seasons were also reported in influenza activity. 10r study showed that clinically RSV was associated with breathing difficulty and pre-existing lung disease, including asthma.This is because RSV causes infections of the lungs and respiratory tract.RSV infection can spread to the lower respiratory tract, causing pneumonia or bronchiolitis (inflammation of the small airway passages entering the lungs).It is so common that most children have been infected with the virus by age 2. In adults, RSV symptoms are mild, however, RSV can cause severe infection in some people, including babies 12 months and younger (infants), especially premature infants, older adults, people with heart and lung disease, or anyone with a weak immune system (immunocompromised). 27r findings showed RSV prevalence (18.5%) among SARI patients were more compared with influenza (13.4%) and ORV (2.8%), which is similar to other countries in the Southeast Asian region including India, 28 Thailand, 29 Bangladesh, 30 Indonesia, 31 and Malaysia 32 ; however, one study from India found influenza (12.7%) was detected more than RSV (8.2%) in SARI patients. 33According to one study, the reported RSV positivity rates in tropical Asian countries range from 9% to 50% depending on the geographic location and sample population. 34 this study, 86% of RSV-associated SARI admissions were below 5 years of age, and more among children aged 0-6 (Adj OR: 3.03; 95% CI: 1.7-5.39),7-23 (Adj OR: 3.01; 95% CI: 1.77-5.12),and 24-59 months (Adj OR: 1.87; 95% CI: 1.05-3.34)as compared with children aged 5-15 years.6][37][38] In addition, research studies show preterm infants have a higher risk for RSV infection, 37 with other risk factors including poor feeding, vomiting, and the rainy season. 32Given maternal administration of RSV vaccines during pregnancy are promising strategy to prevent and reduce RSV infection in newborns, the data in this study may support future investigation of these strategies in Bhutan. 39,40proved understanding of RSV seasonality at the national level is important to ensure the optimal timing of prevention and control measures.Our study showed that RSV was observed throughout the year in 2016 and 2018; however, the peak season occurred in the winter season (January to April) and Monsoon season (June to September) in both years, which is consistent with previous studies and WHO findings. 15,413][44] One study found that RSV seasonality is closely associated with the latitude of the country, 45 and although Bhutan is a small country that experiences four different seasons 46 and falls partly in the subtropical climate, it was recently mapped in the Northern hemisphere by the WHO.

| Strength and limitation
The strength of this study is we included all age groups while other studies mainly focused on pediatric age groups.However, there are few limitations that affect our result analysis.First, our study has not adopted an extended SARI case definition to capture the RSV cases.
Second, outcome status is not known for nearly 40% of participants due to incomplete information during data collection.Third, we did not have additional years of laboratory data of 2017 due to a shortage of supply of laboratory reagents limiting testing capacity.Likewise, RSV typing and gene sequencing were not performed due to a lack of necessary supplies.Lastly, other than medical comorbidities, other risk factors such as preterm and poor feeding were not assessed as the information was not captured by a routine surveillance questionnaire.

| CONCLUSION
Respiratory viruses were detected in a substantial proportion of SARI hospitalizations in Bhutan with RSV detected in nearly one in five of all SARI cases.The high prevalence of RSV in hospitalized SARI infants underscores the importance of RSV prevention, which will soon be available with maternal vaccine or infant monoclonal antibodies (mAb).Therefore, surveillance for RSV is necessary to inform clinical management of SARI, particularly in children, and to implement programmatic preventative and control measures including RSV vaccination.
Patients of all age groups hospitalized with SARI were included for respiratory specimen collection in the years 2016 and 2018 as per the SARI case definition: acute respiratory infection with a history of fever or measured fever of ≥38 C and cough with onset within the last 10 days and requiring hospitalization as prescribed in the ILI and SARI F I G U R E 1 ILI and SARI sentinel surveillance hospitals.
Descriptive statistics were used to analyze the proportion of RSV, influenza, and ORV-associated SARI in different subgroups.Multivariable logistic regression adjusting for age and sex was used to assess the association of RSV with demographic and clinical characteristics.All data were analyzed using STATA 16.1.QGIS 3.16 was used to F I G U R E 2 Algorithm for testing the specimen collected.generate the mapping distribution of the RSV and influenza positivity in the country.

F I G U R E 3
Monthly distribution of viral detection among SARI cases in the year 2016 and 2018.F I G U R E 4 RSV positivity compared with influenza and other respiratory viruses in sentinel sites for all participants.

1
Characteristics of SARI patients admitted to sentinel hospitals in Bhutan in 2016 and 2018.
11,48T A B L E 2 Demographic and clinical characteristics of severe acute respiratory infection positive for RSV compared with those negative for RSV in 2016 and 2018.RSV and influenza were detected from all the SARI sentinel hospitals, but RSV positivity was high in JDWNRH, Thimphu, and Paro Hospital with 26% to 29.6% followed by Trongsa Hospital and Samtse Hospital with 17.3% to 26.0%.As JDWNRH is the only national referral hospital in the country, many people avail medical services including maternal and child health services.11Further, Thimpbeing the capital city, around one fourth of the population resides in Thim-